1. Field of the Invention
This invention relates to fluorometric apparatus, and, more particularly, to apparatus for measuring two different fluorescent substances in a sample.
2. Description of the Prior Art
In its broad sense, "fluorescence" is a phenomenon wherein a substance absorbs ultraviolet rays or other light energy having short wavelength and produces light having a longer wavelength than the absorbed wavelength. A fluorescence microscope is an instrument which utilizes the phenomenon of fluorescence to observe and analyze living cells and other structures.
Nearly all substances are naturally fluorescent to some degree when irradiated with light energy of short wavelength. However, there are certain substances, such as proteins and carbohydrates, which are not fluorescent. Such non-fluorescing substances may be made to fluoresce by dyeing or staining them with a fluorescent substance called a fluorochrome or by employing the fluorescence antibody technique which utilizes the property of antibody molecules of combining with fluorochromes. In some cases, the non-fluorescent substances are stained with two different fluorochromes in the technique known as double staining.
The constituents of samples, such as cells or other structures, which fluoresce either naturally or by the addition of fluorochromes, may be determined by measuring the amount of fluorescent substance contained in the sample. In the technique of fluorescence photometry, for example, the constituents of a sample can be determined by a single metering step when the sample has a single natural fluorescence or after it has been dyed with a single fluorescent substance or fluorochrome. However, in cases where the sample has two different natural fluorescences or where the sample has been stained with two different fluorescent substances or fluorochromes (double staining), it is necessary to employ a highly efficient excitation lamp for irradiating the sample to cause both fluorescences to occur simultaneously and then to measure the emitted light corresponding to each individual fluorescence. Unfortunately, this measurement technique is not desirable for preventing fading of the sample. Consequently, each of the measuring steps must be done very quickly, making measurement extremely difficult. Thus, in order to avoid making errors in the measurements, the operator must have a high degree of skill.
To avoid the need for individually measuring the emitted light corresponding to two simultaneously occurring fluorescences, two excitation lamps may be employed to separately irradiate the sample to cause the fluorescences to occur at different times. If the sample were irradiated by both lamps simultaneously, it would be difficult to distinguish the light emitted during the fluorescences from the light produced by the excitation lamps. For example, in some cases, the wavelength of the light emitted during one or both of the fluorescences may be very close to the wavelength of one of the excitation lamps so that it is difficult to separate the light of the excitation lamp from the emitted light. Thus, the light from the excitation lamp, which is stronger than the emitted light, would mix into the viewing system and the measuring system, making it impossible either to view or to measure the intensities of the two fluorescences.